Skip to main content
Log in

Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc

Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Activation of E2F transcription factors at the G1-to-S phase boundary, with the resultant expression of genes needed for DNA synthesis and S-phase, is due to phosphorylation of the retinoblastoma-related (RBR) protein by cyclin D-dependent kinase (CYCD-CDK), particularly CYCD3-CDKA. Arabidopsis has three canonical E2F genes, of which E2Fa and E2Fb are proposed to encode transcriptional activators and E2Fc a repressor. Previous studies have identified genes regulated in response to high-level constitutive expression of E2Fa and of CYCD3;1, but such plants display significant phenotypic abnormalities. We have sought to identify targets that show responses to lower level induced changes in abundance of these cell cycle regulators. Expression of E2Fa, E2Fc or CYCD3;1 was induced using dexamethasone and the effects analysed using microarrays in a time course allowing short and longer term effects to be observed. Overlap between CYCD3;1 and E2Fa modulated genes substantiates their action in a common pathway with a key role in controlling the G1/S transition, with additional targets for CYCD3;1 in chromatin modification and for E2Fa in cell wall biogenesis and development. E2Fc induction led primarily to gene downregulation, but did not antagonise E2Fa action and hence E2Fc appears to function outside the CYCD3-RBR pathway, does not have a direct effect on cell cycle genes, and promoter analysis suggests a distinct binding site preference.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

References

  • Beemster GTS, De Vusser K, De Tavernier E, De Bock K, Inzé D (2002) Variation in growth rate between Arabidopsis ecotypes is correlated with cell division and A-type cyclin-dependent kinase activity. Plant Physiol 129:854–864

    Article  CAS  PubMed  Google Scholar 

  • Böhner S, Gatz C (2001) Characterisation of novel target promoters for the dexamethasone-inducible/tetracycline-repressible regulator TGV using luciferase and isopentenyl transferase as sensitive reporter genes. Mol Gen Genet 264:860–870

    Article  PubMed  Google Scholar 

  • Botz J, Zerfass-Thome K, Spitkovsky D, Delius H, Vogt B, Eilers M, Hatzigeorgiou A, Jansen-Dürr P (1996) Cell cycle regulation of murine cyclin E gene depends on an E2F binding site in the promoter. Mol Cell Biol 16:3401–3409

    CAS  PubMed  Google Scholar 

  • Bracken AP, Ciro M, Cocito A, Helin K (2004) E2F target genes: unraveling the biology. Trends Biochem Sci 29:409–417

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Connell-Crowley L, Harper JW, Goodrich DW (1997) Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. Mol Biol Cell 8:287–301

    CAS  PubMed  Google Scholar 

  • de Jager SM, Menges M, Bauer U-M, Murray JAH (2001) Arabidopsis E2F1 binds a sequence present in the promoter of S-phase-regulated gene AtCDC6 and is a member of a multigene family with differential activities. Plant Mol Biol 47:555–568

    Article  PubMed  Google Scholar 

  • de Jager SM, Maughan S, Dewitte W, Scofield S, Murray JAH (2005) The developmental context of cell-cycle control in plants. Semin Cell Dev Biol 16:385–396

    Article  PubMed  Google Scholar 

  • de Veylder L, Beeckman T, Beemster GTS, de Almeida Engler J, Ormenese S, Maes S, Naudts M, Van Der Schueren E, Jacqmard A, Engler G, Inzé D (2002) Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor. EMBO J 21:1360–1368

    Article  PubMed  Google Scholar 

  • DeGregori J, Kowalik T, Nevins JR (1995) Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol 15:4215–4224

    CAS  PubMed  Google Scholar 

  • del Pozo JC, Boniotti MB, Gutierrez C (2002) Arabidopsis E2Fc functions in cell division and is degraded by the ubiquitin-SCFAtSKP2 pathway in response to light. Plant Cell 14:3057–3071

    Article  CAS  PubMed  Google Scholar 

  • del Pozo JC, Diaz-Trivino S, Cisneros N, Gutierrez C (2006) The balance between cell division and endoreduplication depends on E2FC-DPB, transcription factors regulated by the ubiquitin-SCFSKP2A pathway in Arabidopsis. Plant Cell 18:2224–2235

    Article  CAS  PubMed  Google Scholar 

  • Dewitte W, Riou-Khamlichi C, Scofield S, Healy JMS, Jacqmard A, Kilby NJ, Murray JAH (2003) Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CycD3. Plant Cell 15:79–92

    Article  CAS  PubMed  Google Scholar 

  • Dewitte W, Scofield S, Alcasabas AA, Maughan SC, Menges M, Braun N, Collins C, Nieuwland J, Prinsen E, Sundaresan V, Murray JAH (2007) Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses. Proc Natl Acad Sci USA 104:14537–14542

    Article  CAS  PubMed  Google Scholar 

  • Dimova DK, Dyson NJ (2005) The E2F transcriptional network: old acquaintances with new faces. Oncogene 24:2810–2826

    Article  CAS  PubMed  Google Scholar 

  • Dimova DK, Stevaux O, Frolov MV, Dyson NJ (2003) Cell cycle-dependent and cell cycle-independent control of transcription by the Drosophila E2F/RB pathway. Genes Dev 17:2308–2320

    Article  CAS  PubMed  Google Scholar 

  • Francis D (2007) The plant cell cycle–15 years on. New Phytol 174:261–278

    Article  CAS  PubMed  Google Scholar 

  • Giangrande PH, Hallstrom TC, Tunyaplin C, Calame K, Nevins JR (2003) Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription factor. Mol Cell Biol 23:3707–3720

    Article  CAS  PubMed  Google Scholar 

  • Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122–2127

    Article  CAS  PubMed  Google Scholar 

  • He SS, Liu J, Xie Z, O’Neill D, Dotson S (2004) Arabidopsis E2Fa plays a bimodal role in regulating cell division and cell growth. Plant Mol Biol 56:171–184

    Article  CAS  PubMed  Google Scholar 

  • Ishida S, Huang E, Zuzan H, Spang R, Leone G, West M, Nevins JR (2001) Role for E2F in control of both DNA replication and mitotic function as revealed from DNA microarray analysis. Mol Cell Biol 21:4684–4699

    Article  CAS  PubMed  Google Scholar 

  • Kosugi S, Ohashi Y (2002a) E2Ls, E2F-like repressors of Arabidopsis that bind to E2F sites in a monomeric form. J Biol Chem 277:16553–16558

    Article  CAS  PubMed  Google Scholar 

  • Kosugi S, Ohashi Y (2002b) Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation. Plant Physiol 128:833–843

    Article  CAS  PubMed  Google Scholar 

  • Kosugi S, Ohashi Y (2003) Constitutive E2F expression in tobacco plants exhibits altered cell cycle control and morphological change in a cell type-specific manner. Plant Physiol 132:2012–2022

    Article  CAS  PubMed  Google Scholar 

  • Lammens T, Boudolf V, Kheibarshekan L, Panagiotis Zalmas L, Gaamouche T, Maes S, Vanstraelen M, Kondorosi E, La Thangue NB, Govaerts W, Inzé D, de Veylder L (2008) Atypical E2F activity restrains APC/CCCS52A2 function obligatory for endocycle onset. Proc Natl Acad Sci USA 10:14721–14726

    Article  Google Scholar 

  • Leone G, DeGregori J, Yan Z, Jakoi L, Ishida S, Williams RS, Nevins JR (1998) E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. Genes Dev 12:2120–2130

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Luo RX, Postigo AA, Dean DC (1998) Rb interacts with histone deacetylase to repress transcription. Cell 92:463–473

    Article  CAS  PubMed  Google Scholar 

  • Magyar Z, Atanassova A, De Veylder L, Rombauts S, Inze D (2000) Characterization of two distinct DP-related genes from Arabidopsis thaliana. FEBS Lett 486:79–87

    Article  CAS  PubMed  Google Scholar 

  • Magyar Z, De Veylder L, Atanassova A, Bakó L, Inzé D, Bögre L (2005) The role of the Arabidopsis E2FB transcription factor in regulating auxin-dependent cell division. Plant Cell 17:2527–2541

    Article  CAS  PubMed  Google Scholar 

  • Mariconti L, Pellegrini B, Cantoni R, Stevens R, Bergounioux C, Cella R, Albani D (2002) The E2F family of transcription factors from Arabidopsis thaliana. J Biol Chem 277:9911–9919

    Article  CAS  PubMed  Google Scholar 

  • Menges M, Hennig L, Gruissem W, Murray JAH (2002) Cell cycle-regulated gene expression in Arabidopsis. J Biol Chem 277:41987–42002

    Article  CAS  PubMed  Google Scholar 

  • Menges M, de Jager SM, Gruissem W, Murray JAH (2005) Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control. Plant J 41:546–566

    Article  CAS  PubMed  Google Scholar 

  • Menges M, Samland AK, Planchais S, Murray JAH (2006) The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis. Plant Cell 18:893–906

    Article  CAS  PubMed  Google Scholar 

  • Miele A, Braastad CD, Holmes WF, Mitra P, Medina R, Xie R, Zaidi SK, Ye X, Wei Y, Harper JW, van Wijnen AJ, Stein JL, Stein GS (2005) HiNF-P directly links the cyclin E/CDK2/p220NPAT pathway to histone H4 gene regulation at the G1/S phase cell cycle transition. Mol Cell Biol 25:6140–6153

    Article  CAS  PubMed  Google Scholar 

  • Müller H, Bracken AP, Vernell R, Moroni MC, Christians F, Grassilli E, Prosperini E, Vigo E, Oliner JD, Helin K (2001) E2Fs regulate the expression of the genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev 15:267–285

    Article  PubMed  Google Scholar 

  • Naouar N, Vandepoele K, Lammens T, Casneuf T, Zeller G, van Hummelen P, Weigel D, Ratsch G, Inze D, Kuiper M, De Veylder L, Vuylsteke M (2009) Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes. Plant J 57:184–194

    Article  CAS  PubMed  Google Scholar 

  • Ohtani K, DeGregori J, Leone G, Herendeen DR, Kelly TJ, Nevins JR (1996) Expression of HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor. Mol Cell Biol 16:6977–6984

    CAS  PubMed  Google Scholar 

  • Ramirez-Parra E, Fründt C, Gutierrez C (2003) A genome-wide identification of E2F-regulated genes in Arabidopsis. Plant J 33:801–811

    Article  CAS  PubMed  Google Scholar 

  • Ramirez-Parra E, López-Matas MA, Fründt C, Gutierrez C (2004) Role of an atypical E2F transcription factor in the control of Arabidopsis cell growth and differentiation. Plant Cell 16:2350–2363

    Article  CAS  PubMed  Google Scholar 

  • Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young R, Aand Dynlacht BD (2002) E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints. Genes Dev 16:245–256

    Article  CAS  PubMed  Google Scholar 

  • Rossignol P, Stevens R, Perennes C, Jasinski S, Cella R, Tremousaygue D, Bergounioux C (2002) AtE2F-a and AtDP-a, members of the E2F family of transcription factors, induce Arabidopsis leaf cells to re-enter S phase. Mol Genet Genomics 266:995–1003

    Article  CAS  PubMed  Google Scholar 

  • Schlisio S, Halperin T, Vidal M, Nevins JR (2002) Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function. EMBO J 21:5775–5786

    Article  CAS  PubMed  Google Scholar 

  • Schnittger A, Schöbinger U, Bouyer D, Weinl C, Stierhof Y-D, Hulskamp M (2002) Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes. Proc Natl Acad Sci USA 99:6410–6415

    Article  CAS  PubMed  Google Scholar 

  • Scofield S, Dewitte W, Murray JAH (2007) The KNOX gene SHOOT MERISTEMLESS is required for the development of reproductive meristematic tissues in Arabidopsis. Plant J 50:767–781

    Article  CAS  PubMed  Google Scholar 

  • Sozzani R, Maggio C, Varotto S, Canova S, Bergounioux C, Albani D, Cella R (2006) Interplay between Arabidopsis activating factors E2Fb and E2Fa in cell cycle progression and development. Plant Physiol 140:1355–1366

    Article  CAS  PubMed  Google Scholar 

  • Tao Y, Kassatly RF, Cress D, Horowitz JM (1997) Subunit composition determines E2F DNA-binding site specificity. Mol Cell Biol 17:6994–7007

    CAS  PubMed  Google Scholar 

  • Tommasi S, Pfeifer GP (1997) Constitutive protection of E2F recognition sequences in the human thymidine kinase promoter during cell cycle progression. J Biol Chem 272:30483–30490

    Article  CAS  PubMed  Google Scholar 

  • Vandepoele K, Vlieghe K, Florquin K, Hennig L, Beemster GTS, Gruissem W, Van de Peer Y, Inzé D, De Veylder L (2005) Genome-wide identification of potential plant E2F target genes. Plant Physiol 139:316–328

    Article  CAS  PubMed  Google Scholar 

  • Vlieghe K, Vuylsteke M, Florquin K, Rombauts S, Maes S, Ormenese S, Van Hummelen P, Van de Peer Y, Inzé D, De Veylder L (2003) Microarray analysis of E2Fa-DPa-overexpressing plants uncovers a cross-talking genetic network between DNA replication and nitrogen assimilation. J Cell Sci 116:4249–4259

    Article  CAS  PubMed  Google Scholar 

  • Vlieghe K, Booudolf V, Beemster GTS, Maes S, Magyar Z, Atanassova A, de Almeida Engler J, De Groodt R, Inzé D, De Veylder L (2005) The DP-E2F-like gene DEL1 controls the endocycle in Arabidopsis thaliana. Curr Biol 15:59–63

    Article  CAS  PubMed  Google Scholar 

  • Weinmann AS, Yan PS, Oberley MJ, Huang TH-M, Farnham PJ (2002) Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis. Genes Dev 16:235–244

    Article  CAS  PubMed  Google Scholar 

  • Weintraub SJ, Prater CA, Dean DC (1992) Retinoblastoma protein switches the E2F site from positive to negative element. Nature 358:259–261

    Article  CAS  PubMed  Google Scholar 

  • Wells J, Boyd KE, Fry CJ, Bartley SM, Farnham PJ (2000) Target gene specificity of E2F and pocket protein family members in living cells. Mol Cell Biol 20:5797–5807

    Article  CAS  PubMed  Google Scholar 

  • Wen H, Andrejka L, Ashton J, Karess R, Lipsick JS (2008) Epigenetic regulation of gene expression by Drosophila Myb and E2F2-RBF via the Myb-MuvB/dREAM complex. Genes Dev 22:601–614

    Article  CAS  PubMed  Google Scholar 

  • Wu F, Lee AS (2002) CDP and AP-2 mediated repression mechanism of the replication-dependent hamster histoneH3.2 promoter. J Cell Biochem 84:699–707

    Article  PubMed  Google Scholar 

  • Yan T, Yoo D, Berardini TZ, Mueller LA, Weems DC, Weng S, Cherry JM, Rhee SY (2005) PatMatch: a program for finding patterns in peptide and nucleotide sequences. Nucleic Acids Res 33:W262–W266

    Article  CAS  PubMed  Google Scholar 

  • Zheng N, Fraenkel E, Pabo CO, Pavletich NP (1999) Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP. Genes Dev 13:666–674

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded by the BBSRC, Cambridge Commonwealth Trust and the European Research Area in Plant Genomics (ERA-PG) project “Plant Stem Cell Network”.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to James A. H. Murray.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11103_2009_9527_MOESM1_ESM.xls

Table S1: CYCD3;1_All. Pairwise comparison data of microarray analysis of dex induction of CYCD3;1 for 6 h, 24 h and 14 days compared to the empty vector control line treated with dex for the same time periods. Column headings and their description are provided in a separate text document Table S1_columnheadings (XLS 2,473 kb)

Supplementary material 14 (DOC 47 kb)

11103_2009_9527_MOESM3_ESM.xls

Table S2: E2Fa/DPa_All. Pairwise comparison data of microarray analysis of dex induction of E2Fa/DPa for 6 h, 24 h and 14 days compared to the empty vector control line treated with dex for the same time periods. Column headings and their description are provided in a separate text document Table S2 columnheadings (XLS 2,888 kb)

Supplementary material 15 (DOC 50 kb)

11103_2009_9527_MOESM5_ESM.xls

Table S3: E2Fc/DPa_All. Pairwise comparison data of microarray analysis of dex induction of E2Fc/DPa for 6 h, 24 h and 14 days compared to the empty vector control line treated with dex for the same time periods. Column headings and their description are provided in a separate text document Table S3 columnheadings(XLS 1,629 kb)

Supplementary material 16 (DOC 50 kb)

11103_2009_9527_MOESM7_ESM.xls

Table S4: CYCD3;1 overlaps. Genes robustly modulated after dex induction of CYCD3;1 for 6 h, 24 h and 14 days which show overlap between any of the three time points are listed. Whether the gene is called increased (I) or decreased (D) is given for each of the three time points, together with the MIPS code and gene description (XLS 48 kb)

11103_2009_9527_MOESM8_ESM.xls

Table S5: E2Fa/DPa overlaps. Genes robustly modulated after dex induction of E2Fa/DPa for 6 h, 24 h and 14 days which show overlap between any of the three time points are listed. Whether the gene is called increased (I) or decreased (D) is given for each of the three time points, together with the MIPS code and gene description (XLS 57 kb)

11103_2009_9527_MOESM9_ESM.xls

Table S6: E2Fc/DPa overlaps. Genes robustly modulated after dex induction of E2Fc/DPa for 6 h,24 h and 14 d which show overlap between any of the three time points are listed. Whether the gene is called increased (I) or decreased (D) is given for each of the three time points, together with the MIPS code and gene description (XLS 38 kb)

11103_2009_9527_MOESM10_ESM.xls

Table S7: 6 h overlaps. Genes robustly modulated after dex induction of CYCD3;1, E2Fa/DPa and E2Fc/DPa for 6 h which show overlap between any of the three transgenes are listed. Whether the gene is called increased (I) or decreased (D) is given for each of the three transgenes, together with the MIPS code and gene description (XLS 26 kb)

11103_2009_9527_MOESM11_ESM.xls

Table S8: 24 h overlaps. Genes robustly modulated after dex induction of CYCD3;1, E2Fa/DPa and E2Fc/DPa for 24 h which show overlap between any of the three transgenes are listed. Whether the gene is called increased (I) or decreased (D) is given for each of the three transgenes, together with the MIPS code and gene description (XLS 171 kb)

11103_2009_9527_MOESM12_ESM.xls

Table S9: 14 d Overlaps. Genes robustly modulated after dex induction of CYCD3;1, E2Fa/DPa and E2Fc/DPa for 14 d which show overlap between any of the three transgenes are listed. Whether the gene is called increased (I) or decreased (D) is given for each of the three transgenes, together with the MIPS code and gene description (XLS 91 kb)

11103_2009_9527_MOESM13_ESM.xls

Table S10: NNNSSCGSSNNN. Robustly modulated genes (reduced dataset) after dex induction of CYCD3;1, E2Fa/DPa and E2Fc/DPa transgenes for 6 h, 24 h and 14 d which have the palindromic E2F-site NNNSSCGSSNNN (where N = A/T/C/G and S = C/G) in the first 500 bp of promoter sequence are listed. The number of sites per promoter, the start and end of the sequence, and the sequence are given (XLS 147 kb)

11103_2009_9527_MOESM14_ESM.xls

Table S11: NNNSSCGS. Robustly modulated genes (reduced dataset) after dex induction of CYCD3;1, E2Fa/DPa and E2Fc/DPa transgenes for 6 h, 24 h and 14 d which have the minimal E2F-site NNNSSCGS (where N = A/T/C/G and S = C/G) in the first 500 bp of promoter sequence are listed. The number of sites per promoter, the start and end of the sequence, and the sequence are given. The distribution of the number of gene promoters with core E2F sites and the number of sites analysed as related to the number of sites per promoter is shown (XLS 316 kb)

11103_2009_9527_MOESM15_ESM.xls

Table S12: All_E2F/DPa_arrays. Comparison of genes robustly modulated after dex induction of E2Fa/DPa for 6 h, 24 h and 14 d and as well as in previously published cDNA and micro-array data (Vlieghe et al. 2003; Vandepoele et al. 2005, Naouar et al. 2009). Genes called increased (I) in at least one of the three timepoints as well as reported increased in at least one of the previous published datasets are listed. The presence of E2F sites of the sequences TTTSSCGS, NNNSSCGS and NNNSSCGSSNNN (where N = A/T/C/G and S = C/G) in the 500 bp upstream sequence is indicated (XLS 35 kb)

11103_2009_9527_MOESM16_ESM.xls

Table S13: Modulated Candidates. Genes not robustly modulated after dex induction of CYCD3;1, E2Fa/DPa and E2Fc/DPa for 6 h, 24 h and 14 d, but that are “modulation candidates” since they meet more relaxed criteria as defined in section "Materials and methods" are listed. Column headings and their description are provided in a separate text document Table S13 columnheadings (XLS 2,849 kb)

Supplementary material 17 (DOC 75 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Jager, S.M., Scofield, S., Huntley, R.P. et al. Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc. Plant Mol Biol 71, 345 (2009). https://doi.org/10.1007/s11103-009-9527-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11103-009-9527-5

Keywords

Navigation